Validation of an IFNγ/IL2 FluoroSpot assay for clinical trial monitoring

Table 3 Inter-day variability of IFNγ, IL2, IFNγ + IL2 responses in the IFNγ/IL2 FluoroSpot assay

Donor	Antigen	Day 1	Day 2	Day 3	Mean	SD	%CV
A
S09	BZLF1	191	145	149	162	25.48	15.76
S11	BZLF1	326	344	351	340	12.90	3.79
S14	BZLF1	223	161	175	186	32.52	17.45
S18	BZLF1	257	194	189	213	37.90	17.77
S10	EBNA3A	27	27	19	24	4.62	18.98
S12	EBNA3A	92	44	35	57	30.64	53.76
S19	EBNA3A	144	127	67	113	40.45	35.90
S15	PHA-L	304	358	311	324	29.37	9.05
S25	PHA-L	77	88	60	75	14.11	18.81
S26	PHA-L	214	242	297	251	42.23	16.82
Mean						27.02	20.81
B
S09	BZLF1	69	62	67	66	3.61	5.46
S11	BZLF1	18	20	17	18	1.53	8.33
S14	BZLF1	48	57	62	56	7.09	12.74
S18	BZLF1	27	29	48	35	11.59	33.43
S10	EBNA3A	17	18	15	17	1.53	9.17
S12	EBNA3A	33	34	29	32	2.65	8.27
S19	EBNA3A	17	12	20	16	4.04	24.74
S15	PHA-L	492	615	572	560	62.42	11.15
S25	PHA-L	344	359	348	350	7.77	2.22
S26	PHA-L	521	617	662	600	72.02	12.00
Mean						17.42	12.75
C
S09	BZLF1	69	56	63	63	6.51	10.38
S11	BZLF1	20	19	18	19	1.00	5.26
S14	BZLF1	54	60	54	56	3.46	6.19
S18	BZLF1	32	32	42	35	5.77	16.34
S10	EBNA3A	14	13	13	13	0.58	4.33
S12	EBNA3A	19	26	20	22	3.79	17.47
S19	EBNA3A	12	11	14	12	1.53	12.39
S15	PHA-L	215	276	258	250	31.34	12.55
S25	PHA-L	50	59	47	52	6.24	12.01
S26	PHA-L	95	130	155	127	30.14	23.79
Mean						9.04	12.07

Values represent the mean number of antigen-specific IFNγ (A), IL2 (B) and IFNγ + IL2 (C) SFC/2 × 10⁵ PBMC (stimulated with 1 µg/ml BZLF1 or EBNA3A peptide pools) or mitogen-specific IFNγ (A), IL2 (B) and IFNγ + IL2 (C) SFC/2 × 10⁵ PBMC/5 × 10⁴ PBMC (stimulated with 2 µg/ml PHA-L) detected on three different assay plates performed on three consecutive days
SD standard deviation, CV coefficient of variation

ISSN: 1479-5876